Method for the identification of {i streptococcus mutans

ABSTRACT

A method for identification of Streptococcus mutans colonies growing among other oral streptococci on mitis salivarius agar has been developed, wherein colonies of Strep. mutans stain dark pink when sprayed with solutions of mannitol or sorbitol and 2,3,5 triphenyl tetrazolium chloride, while colonies of the other streptococci remain blue.

United States Patent [191 Gold I 51 Sept. 2, 1975 1 1 METHOD FOR THE lNDENTlFICATlON OF STREPTOCOCCUS MUTANS [75] Inventor: Olga G. Gold, Boston, Mass.

Forsyth Dental Infirmary for Children, Boston. Mass.

221 Filed: July 16, 1973 211 App1.No.:379,480

[73] Assignee:

[52] US. Cl 1. 195/1035 R; 195/100 OTHER PUBLICATIONS Barnes, E. M., Tetrazolium Reduction as a Means of Differentiating Streptococcus faecalis from Streptococcus fuecium", J. Gen. Microbiol. 14, 57-68, 1956).

Primary E.\un1incrA. Louis Monacell Assismnt E.\'un1inerC. A. Fan Attorney, Agent, (II FirmRichard P. Crowley 1. 1 ABSTRACT A method for identification of Strepmt'occus mummcolonies growing among other oral streptococci on mitis salivarius agar has been developed, wherein c01- onies of Strep. mumns stain dark pink when sprayed with solutions of mannitol or sorbitol and 2,3,5 triphenyl tetrazolium chloride. while colonies of the other streptococci remain blue.

19 Claims, N0 Drawings METHOD FOR THE INDENTIFICATION F STREPTOCOCCUS MUTANS The invention described herein was made in the course of work under a grant or award from the Department of Health. Education and Welfare.

BACKGROUND OF THE INVENTION The identification and enumeration of Streptococcus mustans'in human oral samples are usually based on its distinctive colonial appearance on mitis salviarius agar (Zinner. et al. I965; Krasse. 1966. Jordan. Krasse and Moller. 1968).While this method has been very useful. a simpler procedure for detection of this species would be desirable. since: l recognizing typical and atypical .S'Irepmumns colonies and differentiating them from the other species that grow on the medium requires considerable experience; and (2) the method which necessitates the use of a dissecting microscope can be tedious when large numbers of samples are involved.

SUMMARY OF THE INVENTION This invention relates to a method and solutions for the identification of Streptococcus mumns by the selective fermenting of mannitol or sorbitol by .S'trep.numms in the presence of an indicator.

This invention employs a selective medium for the growth of streptococci. such as mitis salivarius agar or other suitable mediums. The SU'C/LHIHHHIS colonies on the medium are detected among those of other bacteria on the medium by contacting the Slrvpnmmns colonies with sorbitol or preferably mannitol in the presence of an oxidation-reduction indicator which changes color on the selective fermentation of the mannitol or sorbi to] by StrepJnulmzs.

Mannitol or sorbitol. or a combination thereof. must be employed in the practice of the invention. since other polyhydroxyl alcohols and mono saccharides are ineffective in permitting the differentials of Strep/numm' from other organisms. The invention shall be described for the purposes of illustration only by the use of triphenyl tetrazolium chloride (TTC) as the indicator. This indicator in its oxidized state and at a pH of under 6.0 is colorless, while in its reduced state is red. Other indicators which change color or otherwise change in observable or detectable properties on the fermenting of the mannitol may be employed as the indicator. Typically. the method of identification is carried out by contacting; e.g.. spraying. dipping. washing. etc.. the SHC/JJHHIUHS colonies on the medium with mannitol or sorbitol and the indicator at a pH of greater than 6.0; tag, 7 to 8. at a temperature of about to C.

The mannitol or sorbitol may be applied first. followed by the indicator, or where desired. the mannitol or sorbitol and the indicator. such as TTC. may be incorporated in a single solution for application to the .Srrep.mutuns. The solution comprises mannitol or sorbitol. or combinations thereof. in an amount sufficient to permit the S[)'('[7.Hllll(lll.\ to ferment sufficiently mannitol or sorbitol to change the indicator and permit the identification and amount of .S'lrep.nzuruns colonies; e.g.. 0.1 to 20%. Concentrations up to about 10% of the mannitol or sorbitol provide maximum color with a TTC indicator. The solution is a buffered solution at a selected pH; for example. in Sorensons phosphate buffer at a pH of about 7.4. The indicator is present in an amount to provide the desired color change; e.g.

with TTC 0.1 to 5%. A preferred solution comprises up to 10% mannitol or sorbitol. 4'7: 'I'I'C in a buffered phosphate solution at 7.4. Mannitol is the preferred compound. however. sorbitol may also be used. since to .S'rrvpmmruns ferment sorbitol.

A method was developed for the selective staining of .SIru Lmumm colonies which permitted their rapid detection among the unstained colonies of other bacterial species growing on mitis salivarius agar. Srrcpmmmns is one of the few oral streptococcal species capable of fermenting mannitol and sorbitol (Edwardsson. 1968; Carlsson. 1968). The pathway of mannitol and sorbitol catabolism in .S'n'z'pmmuns' has been described by Brown and Wittenberger I973 DESCRIPTION OF THE EMBODIMENTS EXAMPLE I Cells of .S'lri'pmumnx were washed twice and resuspended using Sorensons phosphate buffer pH 7.4 (Documenta Geigy Scientific Tables). These cells incubated in the presence of mannitol and 2. 3. 5 triphenyl tetrazolium chloride (TTC) showed a reduction of the colorless TTC to a red formazan. This reduction was not observed when washed cells of .Sm'plm'm-czis mil/s. .S'rreptm-m'cus .rulii'url'ux or Slrvplm'uccm' .vungm's were used. No reduction of TTC by washed cells of any of the four species was observed in the absence of mannitol. These results indicated that TTC reduction could be used as an indicator of mannitol fermentation by SIIIIIJHHIHILV.

EXAMPLE 2 One representative of each of the 5 serological groups of Strepmzuruns (Bratthall. 1970) were plated on mitis salivarius agar (Difco Laboratories. Detroit. Mich.) and incubated for 24 hrs at 37C under an atmosphere of nitrogen and 5% carbon dioxide fol lowed by overnight aerobic incubation at room temperature. The plates were then sprayed with a 1071 solution of mannitol and incubated aerobically for 3 hrs at 37C. The plates were next sprayed with a 4% solution of 2. 3. 5 triphenyl tctrazolium chloride (Schwarz- Mann. Orangeburg. NY.) and incubated aerobically for another hour at 37C. The solutions of mannitol and TTC were made up in Sorensons phosphate buffer pH 7.4. and sprayed on the plates using glass chromatography spray bottles. The TTC solution was made up in an amber glass bottle just before use. Colonies of Strep.- mumns sprayed in the above manner developed a dark pink color.

EXAMPLE 3 Appropriate dilutions of dental plaque samples were plated. incubated and sprayed with mannitol and TTC as described above for the pure cultures of Strep/nu- !unx. Representatives of colonies that stained pink as well as those that remained blue were subcultured to test their ability to ferment mannitol and sorbitol and to form adhesive growth in sucrose broth. These tests in conjunction with colonial morphology on mitis salivarius agar confirmed the identity of pink colonies as .S'trep.mumns. None of the blue colonies subcultured showed characteristics of Strepwzutuns.

Color contrast between pink and blue colonies was sufficient to allow differential enumeration of Strep.- mutans and the other streptococci with the Quebec colony counter. The pink color of StrepJnuIuns colonies is stable once formed. On prolonged storage of the plates for several hours. a slow non-specific reduction of TTC by the other oral streptococci may result in a feeble staining of the blue colonies thus diminishing the contrast between pink and blue colonies. Colonies from sprayed plates were subcultured at intervals up to 20 hrs to detect viable cells. Positive subscultures could be obtained during this entire period.

Overnight aerobic incubation at room temperature subsequent to 24 hrs anaerobic incubation as used for the identification of SII'L'ILHIHIUHA colonies (Jordan. Krasse, and Moller. 1968) was important for the differential production of formazan by .S'trvpJnutuns. Plates that had been incubated anaerobically could not be immediately sprayed with mannitol and TTC since the overall low Eh caused all colonies to be stained red. The presence of mannitol-fermenting organisms other than SII'LILHIHIUHS in the mouth requires that the differential TTC staining be carried out on a medium selec tive for streptococci.

The only colonies besides those of Stre nmummthat \-\.A'C stained when sprayed with mannitol and TTC were identified as enterococci. This was not unexpected since certain enterococcus species are known to ferment mannitol, and in addition, the ability to reduce TTC has been used to identify strains of enterococci (Barnes. 1956). In the present work a non-specific reduction of TTC by enterococci on mitis salivarius agar was observed even in the absence of mannitol. Formazan stained colonies of the enterococci were round, smooth. flat and bright red and could be easily differentiated from those of StrcpJmiums.

The method described for the differential staining of Strep. mutuns colonies on mitis salivarius agar could be especially useful in studies relating the number of SU'U/LIHIIIUIIS to the total oral streptococcal flora and in general could provide a simple laboratory method for macroscopic identification of SII'UPJIZHIUIIS colonies.

REFERENCES l. Barnes. E. M. 1956. Tetrazolium reduction as a means of differentiating Sn'eptm-m'cus fuccalis from Stn'pmcuccusfiu'cium. J. Gen. Microbiol. 14. 57-68.

2. Bratthall. D. 1970. Demonstration of five serological groups of streptococcal strains resembling Streptococcus mutans. Odont. Revy 21, 143-152.

3. Brown. A. T. and Wittenberger. C. L. 1973. Mannitol and sorbitol catabolism in Streploc'oz'cm' mum/1s. Archs oral Biol. 18. l 17-126.

4. Carlsson. J. 1968. A numerical taxonomic study of human oral streptococci. Odont. Revy 19. 137-160.

5. Documenta Geigy Scientific Tables. 1956. 5th ed. J.

R. Geigy. S. A. Basle. Switzerland, p. 105.

6. Edwardsson, S. 1968. Characteristics of cariesinducing human streptococci resembling Streptococcus IIIHNIIIS. Archs oral Biol. 13. 637-646.

7. Jordan, H. V.. Krasse. B. and Moller, A. 1968. A method of sampling human dental plaque for certain caries inducing" streptococci. Archs oral Biol. 13, 919-927.

8. Krasse, B. 1966. Human streptococci and experimental caries in hamsters. Archs oral Biol. 11, 429-436.

9. Zinncr, D.. Jablon, .l., Aran, A.. and Saslaw, M. 1965. Experimental caries induced in animals by streptococci of human origin. Proc. Soc. Exp. Biol.

What is claimed is:

l. A method of identifying Slrepmcuccus mutans, which method comprises:

a. inoculating a selective growth medium for streptococci with a bacteria sample containing Streptococcm" mutans:

b. incubating the inoculated medium to provide Strqxmutuns colonies;

c. contacting the Strepmzuuuzx with mannitol or sorbitol. or combinations thereof, and fermenting the mannitol or sorbitol in the presence of an oxidation-reduction indicator which changes color on the fermentation of the mannitol or sorbitol by the Strepnzutans'; and

d. identifying the .S'trepJnulans by the differential staining of the Strepwzutuns colonies by change in color of the indicator.

2. The method of claim 1 wherein the selective growth medium is mitis salivarius agar.

3. The method of claim 1 wherein the bacteria sample is a sample from the oral cavity of a patient.

4. The method of claim 1 wherein the indicator is triphenyl tetrazolium chloride.

5. The method of claim 1 wherein the mannitol or sorbitol and the indicator are sprayed onto the medium containing the Strepwmtuns'.

6. The method of claim 5 wherein the medium is first sprayed with an aqueous mannitol or sorbitolcontaining solution and. after incubation. sprayed with a triphenyl tetrazolium chloride solution.

7. The method of claim 1 wherein the mannitol or sorbitol and the indicator are separately applied as so lutions and aerobically incubated after each application.

8. The method of claim 1 wherein incubating the medium is carried out, first. anaerobically, and then aerobically.

9. The method of claim 1 wherein the inoculated incubated stained colonies consist essentially of Strep.- mutarm and enterococci.

10. The method of claim 1 wherein the mannitol or sorbitol and the indicator are applied to the Strep.mutans colonies in an aqueous phosphate buffered solution having a pH of about 7.4.

11. A method of identifying Streptococcus mutans. which method comprises:

a. contacting Strepmutans colonies on a mitis salivarius agar growth medium with mannitol or sorbitol, or combinations thereof, and a triphenyl tetrazolium chloride indicator and fermenting the mannitol or sorbitol by the Strepnrutans and to obtain a change in color of the indicator from colorless to red; and

b. identifying the colonies of StrepJnutans on the agar.

12. The method of claim 11 wherein the growth medium is inoculated with a bacteria sample from the oral cavity of a patient; and wherein the step of identifying includes determining the number of Strepmzutans to the total oral streptococcal flora of the sample.

13. The method of claim 11 wherein the mannitol or sorbitol and the indicator are separately sprayed in aqueous solutions onto the surface of the growth medium, and incubated after each such spraying.

14. The method of claim 11 wherein the mannitol or sorbitol and the triphenyl tetrazolium chloride are in phosphate buffered aqueous solutions having a pH of about 7.4.

15. An aqueous solution useful for the identification of SI)(])I()(U('L'H.\ mamas which comprises in combination:

a. mannitol or sorbitol or a combination thereof: and

h. triphenyl tetrazolium chloride. the solution buffered at a pH of greater than (1.0.

16. The solution of claim which is a phosphate buffered solution having a pH of about 7.4. 7

17. The solution ofclaim 15 wherein the mannitol or sorbitol is present in an amount of up to about (H to 207: by weight. and the triphenyl tetrazolium chloride in an amount of ().l to 5.0% by weight.

18. An aqueous solution useful for the identification of .S'lreplococclis mulmzx which comprises in combination: up to about 10% by weight of mannitol or sorbitol. or combinations thereof. and triphenyl tetrazolium chloride as an indicator. the solution phosphate buffered at a pH of about 7.4.

19. A method of identifying .S'n'epmmluns and differentiating .S'Irupfiiulunt from other streptococci and enterococci. which method comprises:

a. providing a medium containing thereon colonies of SII'U/IJHHIUHS and other organisms. the colonies derived from a sample taken from the oral cavity of a patient;

b. contacting the colonies on the medium with an aqueous solution comprising mannitol or sorbitol or a combination thereof and triphenyl tetra/.olium chloride in the colorless reduced state;

. incubating the colonies so contacted. and fermenting selectively the mannitol or sorbitol by the .S'H'U/LHIHHIHA' to oxidize and change the color of the triphenyl tetrazolium chloride. thereby staining the .S'lre xmumns' colonies a dark pink color; and

d. identifying and differentiating the stained Sire nmmm' colonies from the other organisms on the medium. 

1. A METHOD OF IDENTIFYING STREPTOCOCCUS MUTANS, WHICH METHOD COMPRISES: A. INOCULATING A SELECTIVE GROWTH MEDIUM FOR STRETOCOCCI WITH A BACTERIA SAMPLE CONTAINING STREPTOCOCCUS MUTANS, B. INCUBATING THE INOCULATED MEDIUM TO PROVIDE STREP.MUTANS COLONIES, C. CONTACTING THE STREP.MUTANS WITH MANNITOL OR SORBITOL, OR COMBINATIONS THEREOF, AND FERMENTING THE MANNITOL OR SORBITOL IN THE PRESENCE OF AN OXIDATION-REDUCTION INDICATOR WHICH CHANGES COLOR ON THE FERMENTATION OF THE MANNITOL OR SORBITOL BY THE STREP.MUTANS, AND D. IDENTIFYING THE STREP.MUTANS BY THE DIFFERENTIAL STAINING OF THE STREP.MUTANS COLONIES BY CHANGE IN COLOR OF THE INDICATOR.
 2. The method of claim 1 wherein the selective growth medium is mitis salivarius agar.
 3. The method of claim 1 wherein the bacteria sample is a sample from the oral cavity of a patient.
 4. The method of claim 1 wherein the indicator is triphenyl tetrazolium chloride.
 5. The method of claim 1 wherein the mannitol or sorbitol and the indicator are sprayed onto the medium containing the Strep.mutans.
 6. The method of claim 5 wherein the medium is first sprayed with an aqueous mannitol or sorbitol-containing solution and, after incubation, sprayed with a triphenyl tetrazolium chloride solution.
 7. The method of claim 1 wherein the mannitol or sorbitol and the indicator are separately applied as solutions and aerobically incubated after each application.
 8. The method of claim 1 wherein incubating the medium is carried out, first, anaerobically, and then aerobically.
 9. The method of claim 1 wherein the inoculated incubated stained colonies consist essentially of Strep.mutans and enterococci.
 10. The method of claim 1 wherein the mannitol or sorbitol and the indicator are applied to the Strep.mutans colonies in an aqueous phosphate buffered solution having a pH of about 7.4.
 11. A method of identifying Streptococcus mutans. which method comprises: a. contacting Strep.mutans colonies on a mitis salivarius agar growth medium with mannitol or sorbitol, or combinations thereof, and a triphenyl tetrazolium chloride indicator and fermenting the mannitol or sorbitol by the Strep.mutans and to obtain a change in color of the indicator from colorless to red; and b. identifying the colonies of Strep.mutans on the agar.
 12. The method of claim 11 wherein the growth medium is inoculated with a bacteria sample from the oral cavity of a patient; and wherein the step of identifying includes determining the number of Strep.mutans to the total oral streptococcal flora of the sample.
 13. The method of claim 11 wherein the mannitol or sorbitol and the indicator are separately sprayed in aqueous solutions onto the surface of the growth medium, and incubated after each such spraying.
 14. The method of claim 11 wherein the mannitol or sorbitol and the triphenyl tetrazolium chloride are in phosphate buffered aqueous solutions having a pH of about 7.4.
 15. An aqueous solution useful for the identification of Streptococcus mutans which comprises in combination: a. mannitol or sorbitol or a combination thereof; and b. triphenyl tetrazolium chloride, the solution buffered at a pH of greater than 6.0.
 16. The solution of claim 15 which is a phosphate buffered solution having a pH of about 7.4.
 17. The solution of claim 15 wherein the mannitol or sorbitol is present in an amount of up to about 0.1 to 20% by weight, and the triphenyl tetrazolium chloride in an amount of 0.1 to 5.0% by weight.
 18. An aqueous solution useful for the identification of Streptococcus mutans which comprises in combination: up to about 10% by weight of mannitol or sorbitol, or combinations thereof, and triphenyl tetrazolium chloride as an indicator, the solution phosphate buffered at a pH of about 7.4.
 19. A method of identifying Strep.mutans and differentiating Strep.mutans from other streptococci and enterococci, which method comprises: a. providing a medium containing thereon colonies of Strep.mutans and other organisms, the colonies derived from a sample taken froM the oral cavity of a patient; b. contacting the colonies on the medium with an aqueous solution comprising mannitol or sorbitol or a combination thereof and triphenyl tetrazolium chloride in the colorless reduced state; c. incubating the colonies so contacted, and fermenting selectively the mannitol or sorbitol by the Strep.mutans to oxidize and change the color of the triphenyl tetrazolium chloride, thereby staining the Strep.mutans colonies a dark pink color; and d. identifying and differentiating the stained Strep.mutans colonies from the other organisms on the medium. 